Innate immunity is biogenic initial defense reaction against microbial infection. Microbial components cause various immune responses through recognition by receptors such as Toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-I-like receptors (RLRs), DNA-dependent activator of IFN-regulatory factors (DAI), IFN-γ-inducible protein 16 (IFI16), DDX41, and cyclic GMP-AMP synthase (cGAS).
Toll-like receptors (TLRs) are one-pass transmembrane receptors which form homo dimers or hetero dimers through bonding with ligands, and transmit signals. Human has 10 types of TLRs, and those of which that recognize nucleic acid are TLR3 (recognizes double helical RNA), TLR7 and TLR8 (recognize single-strand RNA), and TLR9 (recognizes unmethylated CpG DNA). TLR3, TLR7, TLR8, and TLR9 are mainly localized in intracellular organelle membranes of ERs and endosomes, and recognize ligands in endosomes, and transmit signals. On the other hand, TLR1 (recognizes triacyl lipoprotein and others), TLR2 (recognizes peptidoglycan and others), TLR4 (recognizes lipopolysaccharide and others), TLR5 (recognizes flagellin and others), and TLR6 (recognizes diacyl lipoprotein and others) which recognize sugars, lipids, and proteins derived from bacteria and viruses are localized on cell surfaces, recognize the microbial surface components and transmit signals on the cell surfaces.
NOD-like receptors (NLRB) are intracellular receptors composing 30 or more large families. They mainly recognize specifically peptidoglycan derived from microorganisms.
RIG-I-like receptors (RLRs) belong to an intracytoplasmic RNA helicase family, and recognize RNAs found in cytoplasm.
DNA-dependent activator of IFN-regulatory factors (DAI), IFN-γ-inducible protein 16 (IFI16), DDX41, cyclic GMP-AMP synthase (cGAS) are identified as cytosolic DNA receptors, but the presence of other cytosolic DNA receptors is suggested.
As described above, various receptors are involved in immunoactivity individually or in collaboration, but the full facts have not been clarified.
Infection, cancer, and allergy are improved by enhancing immunoactivity. For these diseases, research and development for using synthetic oligodeoxynucleotides (hereinafter may be referred to as “ODNs”) containing an unmethylated cytosine-guanine sequence (CpG) as immune-activating medicines have been actively carried out since 2000. The CpG ODNs developed heretofore are largely classified into three classes (class A, class B, and class C).
The CpG-A ODN of class A (also referred to as D type) is a single-stranded ODN which contains CpG in the palindrome sequence with the phosphodiester sugar backbone, and the polyguanine sequence with the phosphorothioated sugar backbone is added to both terminals of the CpG-A ODN (3′ and 5′ terminals) (for example, ODN2216, ODN1585, and D35 shown in Table 1). Two molecules of CpG-A ODN complementarily form a double strand by the palindrome sequence of its phosphodiester sugar backbone, and the polyguanine sequences at both ends form guanine tetramers, so that the two double-stranded CpG-A ODNs are self-assembled to form a tetramer. These tetramers are further self-assembled to double-stranded CpG-A ODN and single-stranded CpG-A ODN molecules, thus forming a higher-order structure (Non-Patent Literature 1). The CpG-A ODN is recognized mainly by TLR9, which are dendritic cells, and induces type-I interferons (IFNs) such as interferon-α (IFN-α) and interferon-β (IFN-β) (Patent Literature 1, and Non-Patent Literature 2).
The CpG-B ODN of class B (also referred to K type) is a single-stranded ODN composed entirely of a phosphorothioated sugar backbone containing CpG (for example, see ODN1826, ODN2006, and K3 shown in Table 1). The CpG-B ODN is mainly recognized by TLR9 of B cells, and induces inflammatory cytokines such as interleukin-6 (IL-6) or IL-12, or tumor necrosis factors (TNFs) (Non-Patent Literature 3). It is known that a complex formed by electrostatically bonding CpG-B ODN with a cationic peptide or cationic liposome induces type-I IFN in the same manner as CpG-A (Non-Patent Literature 4).
The CpG-C ODN of class C is a single-stranded ODN composed entirely of a phosphorothioated sugar backbone containing a palindrome sequence at the 3′ terminal side, and contains CpG at both of the 5′ terminal and 3′ terminal (for example, ODN2395 shown in Table 1). The CpG-C ODN forms a partially double strand from two molecules by its palindrome sequence. The CpG-C ODN exhibits an intermediate nature between CpG-A and CpG-B, and induces both of inflammatory cytokine and type-I IFN (Non-Patent Literature 5).
TABLE 1Table 1. Major CpG DNAs ever developedName(sequenceClassNo)Sequence *)SubjectAODN22165′-ggGGGACGATCGTCgggggg-3′Human(12) AODN1585 5′-ggGGTCAACGTTGAgggggg-3′Mouse(13) AD35 (14)5′-ggTGCATCGATGCAGGGGgg-3′Human, mouse BODN1826 5′-tccatgacgttcctgacgtt-3′Mouse(15) BODN2006 5′-Human(5)tcgtcgttttgtcgttttgtcgtt-3′ BK3 (16)5′-atcgactctcgagcgttctc-3′Human, mouse CODN2395 5′-Human, (17)tcgtcgttttcggcgcgcgccg-3′mouse*) Capital letters represent a phosphodiester sugar backbone, and small letters represent a phosphorothioate sugar backbone; however, all the letters in the sequence listing are written by small letters, and the underline represents a palindrome sequence.